A schematic discussion on Hemolytic Anemia and tools to diagnose and categorize them.

1. Pathogenesis & Lab Diagnosis
of
Sickle Cell Anemia & Thalassemia

2. Pathogenesis
Of
Hemolytic Anemia
Intravascular Extravascular
Abnormal
RBCs
Destruction at
Splenic sinusoids
Release of
hemoglobin Hb transported

3. Fate of Hemoglobin
Porphyrin portion of Heme - Bilirubin
Iron portion of Heme – Bone Marrow stores
Globin – Amino acids recycled
Reduced RBC number
Rebound erythropoiesis
Ineffective Erythropoiesis Extra Medullary Erythropoiesis

4. Pathogenesis
Of
Hemolytic Anemia
Intravascular Extravascular
Abnormal
RBCs
Destruction at
Splenic sinusoids
Release of
hemoglobin
Peripheral Smear
RBC count
Hb%
Decreased
Haptoglobin levels
Hb transported

5. Fate of Hemoglobin
Porphyrin portion of Heme - Bilirubin
Iron portion of Heme – Bone Marrow stores
Globin – Amino acids recycled
Reduced RBC number
Rebound erythropoiesis
Ineffective Erythropoiesis Extra Medullary Erythropoiesis
S. Bilirubin
S. Iron
Hepato splenomegaly
Crew hair cut appearance
BM – M:E ratio reversed
Reticulocyte count

6. Pathogenesis
Of
Hemolytic Anemia
Intravascular Extravascular
Abnormal
RBCs
Destruction at
Splenic sinusoids
Release of
hemoglobin Hb transported
?

7. Abnormal RBCs
Abnormal membrane : Hereditary Spherocytosis
Abnormal Enzymes : G6PD deficiency
Abnormal Hemoglobin
Deficiency
Thalassemia
Heme
Methemoglobinemia
Altered
Sickle Cell Disease
Globin

8. Pathophysiology of Sickle Cell Disease
Robbins & Cotran’s Pathological Basis of Disease

9. Abnormal RBCs
Abnormal membrane : Hereditary Spherocytosis
Abnormal Enzymes : G6PD deficiency
Abnormal Hemoglobin
Deficiency
Thalassemia
Heme
Methemoglobinemia
Altered
Sickle Cell Disease
Globin
Hb Electrophoresis
HPLC
Hb F levels

10. Electrophoretic pattern of Sickle Cell Disease
PJ Russell, iGenetics 3rd ed

11. Functional Assays for sickling
Sickling test:
2% Sodium Metabisulphite reduces oxygen
tension
Induces sickling.
Solubility test:
HbS precipitates in high molarity phosphate
(2.24M) buffer – due to reduced state.

12. Pathophysiology of Thalassemia
Inclusion bodies : β4 inclusion body
Heinz bodies: denatured hemoglobin
Howell Jolly bodies: DNA inclusion body

13. Robbins & Cotran’s Pathological Basis of Disease

14. Electrophoretic pattern of Thalassemia

15. NESTROFT
A screening test for β Thalassemia trait.
Reduced Osmotic Fragility of RBCs.
Normal Osmotic Fragility 0.5 to 0.32%
Negative Positive
DW 0.36% Saline DW 0.36% Saline
Piplani et al., Journal of Clinical and Diagnostic Research, August 2014

16. A word on
Prenatal Diagnosis & Neonatal screening

17. Summary
RBC indices Normal Male Normal female Β Thalassemia
major
Β Thalassemia
trait
Mean
corpuscular
volume (MCV
fl)
89.1±5.01 87.6±5.5 50-70 <79
Mean
corpuscular
hemoglobin
(MCH pg)
30.9±1.9 30.2±2.1 12-20 <27
Hemoglobin
(Hb g/dL)
15.9±1.0 14.0±0.9 <7
Males: 11.5-
15.3
Females: 9.1-
14
Galanello R et al., Hemoglobin. 1979

18. Peripheral Smear: Sickle cells – SCD
Target cells, Heinz bodies,
Inclusion bodies - Thalassemia
Hb Electrophoresis
Hb variants – Hb F in SCD & Thalassemia major
Hb A2 in Thalassemia minor
Sickling tests.
Genetic Mutational Analysis.

19. Thank You!